Very low and low density lipoprotein synthesis and secretion by the human hepatoma cell line Hep-G2: effects of free fatty acid

The liver is a major source of the plasma lipoproteins; however, direct studies of the regulation of lipoprotein synthesis and secretion by human liver are lacking. Dense monolayers of Hep-G2 cells incorporated radiolabeled precursors into protein ([35S]methionine), cholesterol ([3H]mevalonate and [14C]acetate), triacylglycerol, and phospholipid ([3H]glycerol), and secreted them as lipoproteins. In the absence of free fatty acid in the media, the principal lipoprotein secretory product that accumulated had a density maximum of 1.039 g/ml, similar to serum low density lipoprotein (LDL). ApoB-100 represented greater than 95% of the radiolabeled apoprotein of these particles, with only traces of apoproteins A and E present. Inclusion of 0.8 mM oleic acid in the media resulted in a 54% reduction in radiolabeled triacylglycerol in the LDL fraction and a 324% increase in triacylglycerol in the very low density lipoprotein (VLDL) fraction. Similar changes occurred in the secretion of newly synthesized apoB-100. The VLDL contained apoB-100 as well as apoE. In the absence of exogenous free fatty acid, the radiolabeled cholesterol was recovered in both the LDL and the high density lipoprotein (HDL) regions. Oleic acid caused a 50% decrease in HDL radiolabeled cholesterol and increases of radiolabeled cholesterol in VLDL and LDL. In general, less than 15% of the radiolabeled cholesterol was esterified, despite the presence of cholesteryl ester in the cell. Incubation with oleic acid did not cause an increase in the total amount of radiolabeled lipid or protein secreted. We conclude that human liver-derived cells can secrete distinct VLDL and LDL-like particles, and the relative amounts of these lipoproteins are determined, at least in part, by the availability of free fatty acid.     

Structural and compositional changes in very low density lipoprotein triacylglycerols during basal lipolysis.

Triacylglycerols secreted by liver and carried by very low density lipoprotein (VLDL) are hydrolysed in circulation by lipoprotein and hepatic lipases. These enzymes have been shown to have positional and fatty acid specificity in vitro. If there were specificity in basal lipolysis in vivo, triacylglycerol compositions of circulating and newly secreted VLDL would be different. To study this we compared the composition of normal fasting VLDL triacylglycerol of Wistar rats to that obtained after blocking lipolysis by Triton WR1339, which increased plasma VLDL triacylglycerol concentration about 4.7-fold in 2 h. Analyses of molecular species of sn-1,2- and sn-2,3-diacylglycerol moieties and stereospecific triacylglycerol analysis revealed major differences between the groups in the VLDL triacylglycerol composition. In nontreated rats, the proportion of 16:0 was higher and that of 18:2n-6 lower in the sn-1 position. The proportion of 14:0 was lower in all positions and that of 18:0 was lower in the sn-1 and sn-3 positions in nontreated rats whereas the proportions of 20:4n-6, 20:5n-3, 22:5n-3 and 22:6n-3 were higher in the sn-1 and lower in the sn-2 position. These results suggest that the fatty acid of the sn-1 position is the most decisive factor in determining the sensitivity for hydrolysis of the triacylglycerol. In addition, triacylglycerol species with highly unsaturated fatty acids in the sn-2 position also favoured hydrolysis. The in vivo substrate specificity followed only partly that obtained in in vitro studies indicating that the nature of molecular association of fatty acids in natural triacylglycerol affects its susceptibility to lipolysis. To conclude, our results indicate that preferential basal lipolysis leads to major structural differences between circulating and newly secreted VLDL triacylglycerol. These differences extend beyond those anticipated from analysis of total fatty acids and constitute a previously unrecognized feature of VLDL triacylglycerol metabolism.     

Isolation of very low density lipoprotein phospholipids enriched in ethanolamine phospholipids from rats injected with Triton WR 1339

Phospholipids carried by very low density lipoprotein (VLDL) are hydrolysed in circulation by lipoprotein and hepatic lipases and lecithin-cholesterol acyltransferase. We have previously demonstrated [J.J. Agren, A. Ravandi, A. Kuksis, G. Steiner, Structural and compositional changes in very low density lipoprotein triacylglycerols during basal lipolysis, Eur. J. Biochem. 269 (2002) 6223-6232] that the infusion of Triton WR 1339 (TWR), which inhibits these lipases, leads in 2 h to five-fold increase in VLDL triacylglycerol concentration along with major differences in the composition of their molecular species. The present study demonstrates that the accumulation of triacylglycerols is accompanied by major changes in the content of the VLDL phospholipids, of which the most significant is the enrichment of phosphatidylethanolamine (PtdEtn). This finding coincides with the enrichment in PtdEtn demonstrated in the VLDL of a hepatocytic Golgi fraction but it had not been demonstrated that the Golgi VLDL, along with its unusual phospholipid composition, can be directly transferred to plasma. Aside from providing an easy access to nascent plasma VLDL, the TWR infusion demonstrates that lipoprotein and hepatic lipases are also responsible for the degradation of plasma VLDL PtdEtn, as independently demonstrated for plasma phosphatidylcholine. Our results indicate also, with the exception of lysophosphatidylcholine, that preferential basal hydrolysis no dot lead to major differences in molecular species composition between circulating and newly secreted VLDL phospholipids. The comparison of the molecular species composition of VLDL and liver phospholipids suggests a selective secretion of PtdEtn and sphingomyelin molecular species during VLDL secretion.     

Lipoprotein metabolism in hepatic lipase deficiency: studies on the turnover of apolipoprotein B and on the effect of hepatic lipase on high density lipoprotein

Hepatic lipase deficiency produces significant distortion in the plasma lipoprotein profile. Particles with reduced electrophoretic mobility appear in very low density lipoprotein (VLDL). Intermediate density lipoprotein (IDL) increases markedly in the circulation and plasma low density lipoprotein (LDL) levels fall. At the same time there is a mass redistribution within the high density lipoprotein (HDL) spectrum leading to dominance in the less dense HDL2 subfraction. The present study examines apolipoprotein B turnover in a patient with hepatic lipase deficiency. The metabolism of large and small very low density lipoproteins was determined in four control subjects and compared to the pattern seen in the patient. Absence of the enzyme did not affect the rate at which large very low density lipoproteins were converted to smaller particles within this density interval (i.e., of VLDL). However, subsequent transfer of small very low density lipoproteins to intermediate density particles was retarded by 50%, explaining the abnormal accumulation of VLDL in the patient's plasma. Despite this, intermediate density particles accumulated to a level 2.4-times normal because their subsequent conversion to low density lipoprotein has been almost totally inhibited. Consequently, the plasma concentration of low density lipoprotein was only 10% of normal. On the basis of these observations, hepatic lipase appears to be essential for the conversion of small very low density and intermediate density particles to low density lipoproteins. The pathways of direct plasma catabolism of these species were not affected by the enzyme defect. In vitro studies were performed by adding purified hepatic lipase to the patient's plasma.(ABSTRACT TRUNCATED AT 250 WORDS)     

The catabolism of human and rat very low density lipoproteins by perfused rat hearts.

The catabolism of human and rat 125I-labelled very low density lipoproteins (VLDL) was compared by perfusing the lipoproteins through beating rat hearts. Triacylglycerol was removed from the VLDL to a greater extent than the protein moiety, leaving remnants containing relatively more apo-B and less apo-C. The change in apo-C content of the remnants correlated with the loss of triacylglycerol. The extent of removal of triacylglycerol from the rat and human VLDL was similar and in most cases appeared to saturate the heart lipoprotein lipase. The remnants were slightly smaller in size than the VLDL, and included particles which appeared to be partially emptied. In addition to remnants of d less than 1.019 g/ml, iodinated lipoproteins derived from rat and human VLDL were recovered at d 1.019-1.063 and 1.063-1.21 g/ml. The former contained largely cholesterol and cholesteryl esters, while phospholipids were the dominant lipid in the latter. An average of 40% of the 125I-labelled apoprotein lost from the VLDL was associated with the perfused hearts. Very little d 1.019-1.063 g/ml lipoprotein was produced from low (physiological) concentrations of rat VLDL, most of the lipoprotein being removed by the heart. However, lipoproteins of density 1.019-1.063 g/ml were formed from human VLDL at all concentrations in the perfusate, as well as from higher concentrations of the rat VLDL. Agarose gel filtration of lipoproteins following heart perfusion with human VLDL revealed large aggregates containing particles which resemble low density lipoproteins (LDL) in electron microscopic appearance and apoprotein composition, since they contain largely apo-B. These data suggest that at normal concentrations rat VLDL are almost completely catabolised and taken up by the heart without the formation of LDL, while LDL is produced from human VLDL at all concentrations.     

Stimulation by oleic acid of incorporation of L-[4,5-3H]leucine into very low density lipoprotein apoprotein by the isolated perfused rat liver

Livers from normal fed or fasted (24h) rats were perfused in vitro to determine whether fatty acid affects the biosynthesis of very low density lipoprotein (VLDL) apoprotein. Oleate stimulated VLDL triacylglycerol output and increased incorporation of L-[4,5-3H]leucine into VLDL apoprotein in both the fed and fasted groups. The increased incorporation of [3H]leucine was mainly into VLDL-apoprotein E. The total mass of VLDL apoprotein secreted was also stimulated by oleate proportionately. These data suggest that fatty acids may stimulate hepatic synthesis and/or secretion of the VLDL apoproteins and that apo E, may be required for the formation and secretion of triacyl-glycerol in the VLDL.     

Cell triacylglycerol accumulation from very low density lipoproteins isolated from normal and hypertriglyceridemic human sera.

Human fibroblast cells in culture increased their intracellular triacylglycerol levels when exposed to very low density lipoproteins (VLDL) isolated from human plasma. This response was dependent on the amount of VLDL added. VLDL from normal, type IV or type V sera gave similar results. Lipoprotein lipase enhanced this intracellular triacylglycerol accumulation. It was concluded that human fibroblast cells in culture have at least two mechanisms for triacylglycerol uptake from VLDL: (1) uptake from intact lipoprotein either by surface transfer of lipoprotein lipid or internalization of the entire lipoprotein particle, and (2) re-esterification of lower glyceride and fatty acids released by lipoprotein lipase degradation of VLDL.     

Resistance of a very low density lipoprotein subpopulation from familial dysbetalipoproteinemia to in vitro lipolytic conversion to the low density lipoprotein density fraction

In vitro lipolysis of very low density lipoprotein (VLDL) from normolipidemic and familial dysbetalipoproteinemic plasma by purified bovine milk lipoprotein lipase was studied using the combined single vertical spin and vertical autoprofile method of lipoprotein analysis. Lipolysis of normolipidemic plasma supplemented with autologous VLDL resulted in the progressive transformation of VLDL to low density lipoprotein (LDL) via intermediate density lipoprotein (IDL) with the transfer of the excess cholesterol to high density lipoprotein (HDL). At the end of 60 min lipolysis, 92-96% of VLDL triglyceride was hydrolyzed, and, with this process, greater than 95% of the VLDL cholesterol and 125-I-labeled VLDL protein was transferred from the VLDL to the LDL and HDL density region. When VLDL from the plasma of an individual with familial dysbetalipoproteinemia was substituted for VLDL from normolipidemic plasma, less than 50% of the VLDL cholesterol and 65% of 125I-labeled protein was removed from the VLDL density region, although 84-86% of VLDL triglyceride was lipolyzed. Analysis of familial dysbetalipoproteinemic VLDL fractions from pre- and post-lipolyzed plasma showed that the VLDL remaining in the postlipolyzed plasma (lipoprotein lipase-resistant VLDL) was richer in cholesteryl ester and tetramethylurea-insoluble proteins than that from prelipolysis plasma; the major apolipoproteins in the lipoprotein lipase-resistant VLDL were apoB and apoE. During lipolysis of normolipidemic VLDL containing trace amounts of 125I-labeled familial dysbetalipoproteinemic VLDL, removal of VLDL cholesterol was nearly complete from the VLDL density region, while removal of 125I-labeled protein was only partial. A competition study for lipoprotein lipase, comparing normolipidemic and familial dysbetalipoproteinemic VLDL to an artificial substrate ([3H]triolein), revealed that normolipidemic VLDL is clearly better than familial dysbetalipoproteinemic VLDL in competing for the release of 3H-labeled free fatty acids. The results of this study suggest that, in familial dysbetalipoproteinemic individuals, a subpopulation of VLDL rich in cholesteryl ester, apoB, and apoE is resistant to in vitro conversion by lipoprotein lipase to particles having LDL-like density. The presence of this lipoprotein lipase-resistant VLDL in familial dysbetalipoproteinemic subjects likely contributes to the increased level of cholesteryl ester-rich VLDL and IDL in the plasma of these subjects.     

Impaired triacylglycerol catabolism in hypertriglyceridemia of the diabetic, cholesterol-fed rabbit: a possible mechanism for protection from atherosclerosis

The etiology of the hypertriglyceridemia in alloxan-diabetic rabbits was studied by two independent methods. Production and removal rates of VLDL triacylglycerol were measured in diabetic rabbits by injection of [3H]palmitate-labelled VLDL. Similarly, triacylglycerol total removal rates were determined in non-diabetic rabbits which were infused with Intralipid to mimic the plasma triacylglycerol concentrations of diabetic rabbits. Compared to nondiabetic rabbits, triacylglycerol removal rats were decreased in diabetic rabbits, particularly at higher levels of plasma triacylglycerol. During cholesterol and triacylglycerol supplementation of the diet, post-heparin plasma lipoprotein lipase activity of diabetic rabbits with severe hypertriglyceridemia averaged 36% of that of nondiabetics, suggesting an impaired triacylglycerol removal capacity. Furthermore, plasma triacylglycerol was inversely related to post-heparin plasma lipoprotein lipase activity among diabetic rabbits. VLDL triacylglycerol production increased with increasing plasma triacylglycerol concentration among diabetic cholesterol-fed rabbits with moderately severe hypertriglyceridemia, but reached an apparent plateau among rabbits with plasma triacylglycerol concentrations from approx. 2000-9000 mg/dl. Thus, severe hypertriglyceridemia in this model of insulin deficiency can be attributed only partially to VLDL hypersecretion, whereas a removal defect, resulting in saturation of the triacylglycerol removal mechanism, appears to be largely responsible. The impaired removal of plasma triacylglycerol is also related to the presence of cholesterol predominantly in lipoproteins of increased size. The data support the hypothesis that protection against atherosclerosis in cholesterol-fed diabetic rabbits results from exclusion of very large cholesterol-containing lipoproteins from the arterial wall.     

Lipoprotein lipase-mediated sequestration of long-chain polyunsaturated triacylglycerols in serum LDL from normal and hypothyroid rats

Rat serum VLDL, unlike human, contains significant proportions of triacylglycerols with polyunsaturated C20 and C22 fatty acids. Hypothyroidism in this species is characterized by low levels of serum VLDL, the accumulation of LDL, elevated levels of lipoprotein lipase and depressed hepatic lipase activity. The hypothyroid rat thus represents an interesting model in which to study hepatic VLDL metabolism and the substrate specificity of lipoprotein lipase. This report shows that serum IDL and LDL in both euthyroid and hypothyroid rats contain progressively enhanced proportions of triacylglycerols with polyunsaturated C20 and C22 fatty acids when compared to VLDL. Hypothyroidism resulted in a decrease in the proportion of 22:6 fatty acid within the serum VLDL triacylglycerols when compared to euthyroid VLDL. Lipolysis of VLDL from euthyroid rats in vitro using the perfused rat heart system resulted in increases or sequestration of triacylglycerols containing long-chain polyunsaturated fatty acids within the IDL fraction similar to those seen in vivo. It is concluded that lipoprotein lipase-mediated hydrolysis of VLDL triacylglycerols and the conversion of VLDL to IDL and LDL in the rat results in a progressive sequestration of the longer-chain polyunsaturated triacylglycerol molecular species with the IDL and LDL.     

Heterogeneity of very-low-density lipoprotein remnants bound and taken up by liver of starved rat in vivo.

Very-low-density lipoprotein (VLDL), labelled in vivo with [9,10-3H]oleate, was taken up rapidly by liver after injection in vivo. Initially, radioactive lipoprotein remnants in the VLDL density range were present in liver as a bound extracellular pool that could be released by perfusion with polyphosphate or heparin. The bound remnant showed a decrease in mean diameter and an increased proportion of cholesteryl ester as a function of time after injection. When VLDL of different mean diameters was injected, it was found that: (1) total uptake by liver was independent of diameter; (2) small VLDL was not taken up more rapidly than large VLDL; and (3) Large VLDL lost no more triacylglycerol before binding than did small VLDL and larger species of mean diameter greater than 40 nm were bound. It is concluded that there is no unique VLDL remnant taken up by liver in vivo. When livers were perfused after binding radioactive VLDL in vivo, the lipoprotein was metabolized, with the production of water-soluble products, and this metabolism was inhibited by chloroquine.     

Effect of dietary fat composition on the metabolism of triacylglycerol-rich plasma lipoproteins in the postprandial phase in meal-fed rats

Rats conditioned to eating fixed-size meals (meals at 7 AM and 7 PM), consuming diets rich in palm oil or sunflower seed oil, were used to study the metabolism of chylomicrons and hepatic very low density lipoproteins (VLDL) as a function of time after meal consumption. Rats fed a palm oil diet had higher serum triacylglycerol levels at 7 AM, before the meal (1.96 +/- 0.25 mM vs. 1.09 +/- 0.09 mM) and reached higher levels postprandially (4.32 +/- 0.48 mM vs. 2.87 +/- 0.18 mM) than sunflower seed oil-fed animals, due to higher levels of hepatic VLDL (at 7 AM) and higher levels of chylomicrons and hepatic VLDL (in the postprandial phase). These differences in serum triacylglycerol concentrations between the diets tested were found not to be due to differences in hepatic VLDL triacylglycerol secretion (similar rate for both dietary groups and not very much affected by meal consumption) or chylomicron triacylglycerol secretion (similar response profiles on both diets), pointing towards differences in plasma triacylglycerol catabolism. Subsequent double-label studies on triacylglycerol catabolism of chylomicrons from palm oil- and sunflower seed oil-fed animals in chow-fed recipients showed that palm oil triacyglycerol is catabolized slower than sunflower seed oil triacylglycerol. Furthermore, activities of postheparin plasma lipoprotein lipase tended to be higher in sunflower seed oil-fed animals. From these data we conclude that the relative hypertriglyceridemia found in palm oil-fed animals is due to less efficient catabolism and not to increased synthesis of plasma triacylglycerol.     

Effects of threonine-poor apolipoproteins on post-heparin plasma hepatic triacylglycerol lipase and lipoprotein lipase

Whole-irradiated rabbit pre-heparin plasma had an important inhibitory effect on hepatic triacylglycerol lipase and lipoprotein lipase activities, whereas control rabbit pre-heparin plasma slightly inhibited hepatic triacylglycerol lipase activity at a high concentration and enhanced lipoprotein lipase activity. As some apolipoproteins were known to modulate these two lipolytic enzymes, the inhibitory effects of irradiated rabbit plasma were investigated in apolipoproteins. Three apolipoproteins, with isoelectric points of about 6.58, 6.44 and 6.12, characterized by their low content in threonine (threonine-poor apolipoproteins) were produced in high concentrations in rabbit VLDL and HDL after irradiation. The effects of these apolipoproteins on control rabbit post-heparin plasma hepatic triacylglycerol lipase and extrahepatic lipoprotein lipase were studied. Threonine-poor apolipoproteins substantially inhibited the hepatic triacylglycerol lipase activity and enhanced the apolipoprotein C-II-stimulated activity of lipoprotein lipase. The amounts of these apolipoproteins in triacylglycerol-rich lipoprotein particles may determine the lipolytic activity of lipoprotein lipase and hepatic triacylglycerol lipase in triacylglycerol hydrolysis. The existence of another inhibitor of lipoprotein lipase remains to be determined.     

Cholesterol is required for secretion of very-low-density lipoprotein by rat liver.

To study potential effects of hepatic cholesterol concentration on secretion of very-low-density lipoprotein (VLDL) by the liver, male rats were fed on unsupplemented chow, chow with lovastatin (0.1%), or chow with lovastatin (0.1%) and cholesterol (0.1%) for 1 week. Livers were isolated from these animals and perfused in vitro, with a medium containing [2-14C]acetate, bovine serum albumin and glucose in Krebs-Henseleit buffer, and with an oleate-albumin complex. With lovastatin feeding, the hepatic concentrations of cholesteryl esters and triacylglycerols before perfusion were decreased, although free cholesterol was unchanged. However, hepatic secretion of all the VLDL lipids was decreased dramatically by treatment with lovastatin. Although total secretion of VLDL triacylglycerol, phospholipid, cholesterol and cholesteryl esters was decreased, the decrease in triacylglycerol was greater than that in free cholesterol or cholesteryl esters, resulting in secretion of a VLDL particle enriched in sterols relative to triacylglycerol. In separate studies, the uptake of VLDL by livers from control animals or animals treated with lovastatin was measured. Uptake of VLDL was estimated by disappearance of VLDL labelled with [1-14C]oleate in the triacylglycerol moiety, and was observed to be similar in both groups. During perfusion, triacylglycerol accumulated to a greater extent in livers from lovastatin-fed rats than in control animals. The depressed output of VLDL triacylglycerols and the increase in triacylglycerol in the livers from lovastatin-treated animals was indicative of a limitation in the rate of VLDL secretion. Addition of cholesterol (either free cholesterol or human low-density lipoprotein) to the medium perfusing livers from lovastatin-fed rats, or addition of cholesterol to the diet of lovastatin-fed rats, increased the hepatic concentration of cholesteryl esters and the output of VLDL lipids. The concentration of cholesteryl esters in the liver was correlated with the secretion of VLDL by the liver. These data suggest that cholesterol is an obligate component of the VLDL required for its secretion. It is additionally suggested that cholesteryl esters are in rapid equilibrium with a small pool of free cholesterol which comprises a putative metabolic pool available and necessary for the formation and secretion of the VLDL. Furthermore, the specific radioactivity (d.p.m./mumol) of the secreted VLDL free cholesterol was much greater than that of hepatic free cholesterol, suggesting that the putative hepatic metabolic pool is only a minor fraction of total hepatic free cholesterol.     

An apo-E-free very low density lipoprotein enriched in phosphatidylethanolamine in human plasma

Normal human plasma contains a fraction of very low density lipoprotein (VLDL) which, unlike most VLDL, contains no apolipoprotein E and, unlike apo-E-containing VLDL, is enriched in phosphatidylethanolamine. This fraction made up 0.28 +/- 0.09 of total VLDL triglyceride. Interconversion of the two isolated VLDL fractions was not detected during incubation (2 h, 37 degrees C) and they may represent the physical forms of apo-B corresponding to distinct metabolic pathways in plasma.     

Effects of estrogen administration on the lipoproteins and apoproteins of the chicken.

The plasma lipoproteins of estrogen-treated and untreated sexually immature hens have been compared with respect to their concentration in plasma, protein and lipid composition, particle size, and and apoprotein composition. Administration of diethylstilbestrol resulted in a 400-fold rise in the concentration of very low density lipoprotein (VLDL), a 70-fold rise in low density lipoprotein (LDL), and a marked reduction in high density lipoprotein (HDL) protein. It also resulted in the production of LDL and HDL which were enriched in triacylglycerol, while the proportion of cholesterol in all three lipoprotein fractions decreased. In contrast to the lipoproteins from untreated birds, lipoproteins of density less than 1.06 g/ml from estrogen-treated birds were not clearly separable into discrete VLDL and LDL fractions, but appeared to be a single ultracentrifugal class. The apoprotein composition of VLDL and LDL from untreated birds differed from each other; however, the apoprotein patterns of VLDL and LDL from estrogen-treated birds were indistinguishable: both contained a large amount of low molecular weight protein in addition to the high molecular weight component that predominates in the untreated state. The apoprotein composition of HDL was also markedly altered by estrogen administration: the 28,000 mol. wt. protein (apo A-I) decreased in amount from 65% to less than 5% of the total, while a low molecular weight (Mr = 14,000) protein and as yet poorly defined high molecular weight components became predominant. These observations indicate that the hyperlipidemia induced by estrogen administration is accompanied by marked alterations, both qualitative and quantitative, in the plasma lipoproteins.     

Studies on the mechanism of hypertriglyceridemia in Tangier disease. Determination of plasma lipolytic activities, k1 values and apolipoprotein composition of the major lipoprotein density classes

Mechanisms responsible for hypertriglyceridemia in Tangier disease were elucidated by an analysis of the plasma post-heparin lipolytic activities and the structural and metabolic properties of very low (VLDL) and low (LDL) density lipoproteins. The levels of lipoprotein lipase activity in six Tangier patients were significantly lower (P less than 0.001) than in 40 control subjects (8.1 +/- 3.3 (+/- S.D.) vs. 14.1 +/- 3.7 units/ml). In contrast, the levels of hepatic triacylglycerol lipase were higher (P less than 0.01) than in normal controls (14.4 +/- 3.9 vs. 9.3 +/- 4.0 units/ml). Because kinetic parameters such as Km or Vmax cannot be obtained with naturally occurring triacylglycerol-rich lipoproteins, the pseudo-first-order rate constant (k1) of triacylglycerol hydrolysis was used to assess the effectiveness of triacylglycerol-rich lipoproteins as substrates for lipoprotein lipase. The k1 values for Tangier VLDL (k1 = 0.017 +/- 0.002 min-1) were significantly lower (P less than 0.001) than the k1 values (0.036 +/- 0.008 min-1) for control VLDL. Both the Tangier and control LDL2 are similar in their resistance to the action of lipoprotein lipase, as shown by their low k1 values (0.002 +/- 0.001 and 0.001 +/- 0.001 min-1, respectively). The major compositional difference between the lipoproteins of Tangier disease and normal subjects was a significant increase in the percent content of apolipoprotein A-II in all lipoprotein particles with d less than 1.063 g/ml, with the greatest increase occurring in VLDL and the lowest in LDL2. These results were interpreted as indicating that, in Tangier disease, there is a lower reactivity of VLDL with lipoprotein lipase which may in part be attributed to the abnormal apolipoprotein composition. This finding, in conjunction with the reduced levels of lipoprotein lipase activity, may explain the hypertriglyceridemia in Tangier disease.     

Metabolic behavior of hepatic VLDL and plasma LDL apoB-100 in African green monkeys

Recently, evidence has accumulated suggesting that significant amounts of plasma low density lipoproteins (LDL) may be derived by direct production. These plasma very low density lipoprotein (VLDL)-independent sources include the production and secretion of LDL-like particles directly by the liver, and/or a small pool of nascent precursor particles that are converted rapidly to LDL. The current studies were designed to test the hypothesis that hepatic VLDL represent a rapidly turning over precursor pool to plasma LDL in African green monkeys. Livers from African green monkeys were perfused with serum-free medium containing [3H]leucine or 3H-labeled amino acids for 4-6 hr. Hepatic [3H]VLDL and autologous plasma 125I-labeled LDL were injected simultaneously into recipient animals and density gradient ultracentrifugation and gel filtration were used to characterize the distribution of 3H and 125I radioactivity at selected times after injection. These studies show that 4 to 66% of the injected dose of hepatic VLDL [3H]apoB-100 was metabolized extremely rapidly into particles that resembled the recipient's plasma LDL by size and density. Based on the kinetic model developed to describe the metabolic behavior of hepatic VLDL [3H]apoB-100, the estimated maximal pool size of hepatic VLDL apoB-100 in these animals was very small (0.042 and 0.112 mg) and represented, at best, approximately 10% of the average plasma VLDL apoB-100 mass found in cholesterol-fed African green monkeys. In addition, the radiolabeled hepatic LDL appear to be metabolized similarly to plasma LDL. That is, the rapid conversion of hepatic VLDL as well as the direct production of hepatic particles within the LDL density range appear to contribute to plasma LDL. Metabolic heterogeneity was also seen within the LDL class. The more buoyant subfraction (LDL1) had a higher turnover rate than the more dense subfraction (LDL2) and hepatic VLDL-derived [3H]LDL1 had a slower final rate of plasma disappearance than the plasma-derived 125I-labeled LDL1 in most animals. The results from these studies suggest that a small pool of hepatic VLDL can be converted very rapidly to plasma LDL and may contribute significantly to the large plasma pool of LDL seen in cholesterol-fed African green monkeys. This pathway may be analogous to the pathway in some human subjects in which a portion of human plasma VLDL is converted rapidly into LDL without passing through a delipidation cascade, often referred to as direct LDL production.     

Plasma zinc as an index of vitellogenin production and reproductive status in the domestic fowl.

1. A technique is described for the accurate determination of circulating vitellogenin concentrations by measurement of plasma zinc concentrations before and after selective precipitation of lipoproteins in the domestic fowl. 2. Vitellogenin zinc concentrations exhibit a high degree of correlation with those of another major egg yolk precursor, very low density lipoprotein (VLDL) in immature female birds, during developing and maximum egg production in mature birds and following cessation of egg laying. 3. Mature female patterns of plasma vitellogenin and VLDL were induced by injection of oestradiol 17 beta (5 mg/kg body weight per day for 3 days) into adult cockerels. 4. It is suggested that measurement of plasma zinc provides a simple and accurate technique for the estimation of vitellogenin production and reproductive status in the domestic fowl and that this may be applied to other oviparous vertebrates.     

7-ketocholesterol inhibits VLDL secretion by cultured human and rabbit hepatocytes

7-ketocholesterol, one of the major product of autoxidation of dietary cholesterol, was found to inhibit secretion of very low density lipoprotein [14C]cholesterol, [14C]triacylglycerol and [35S]apoprotein B,E,C by cultured human and rabbit hepatocytes. A parallel inhibition (about 35%) of cholesterol synthesis but not of triacylglycerol formation was observed. Incubation with 10 micrograms/ml of oxysterol also reduced the total apo-B secretion measured by ELISA and increased intracellular apo-B mRNA level. These results seem to indicate that 7-ketocholesterol decreases secretion of very low density lipoprotein (VLDL) particles and exerts inhibitory effects on apo-B production at the co-translational or posttranslational level.